Isolation of nucleic acids

ABSTRACT

A method for extracting nucleic acids from a biological material such as blood comprises contacting the mixture with a material at a pH such that the material is positively charged and will bind negatively charged nucleic acids and then eluting the nucleic acids at a pH when the said materials possess a neutral or negative charge to release the nucleic acids. The nucleic acids can be removed under mildly alkaline conditions to the maintain integrity of the nucleic acids and to allow retrieval of the nucleic acids in reagents that are immediately compatible with either storage or analytical testing.

This application is a continuation-in-part of U.S. Ser. No. 09/586,009,filed 2 Jun. 2000, which derives from PCT/GB98/03602, filed 4 Dec. 1998,which claims priority from UK patent application numbers 9725839.6,filed 6 Dec. 1997, and 9815541.9, filed 17 Jul. 1998. The entiredisclosure of the '009 application is incorporated by reference herein.

The present invention relates to a method for extracting nucleic acidsand other biomolecules from biological materials, particularly blood andother liquid samples.

There is a very large demand for DNA analysis for a range of purposesand this has lead to the requirement for quick, safe, high throughputmethods for the isolation and purification of DNA and other nucleicacids.

Samples for use for DNA identification or analysis can be taken from awide range of sources such as biological material such as animal andplant cells, faeces, tissue etc. also samples can be taken from soil,foodstuffs, water etc.

Existing methods for the extraction of DNA include the use ofphenol/chloroform, salting out, the use of chaotropic salts and silicaresins, the use of affinity resins, ion exchange chromatography and theuse of magnetic beads. Methods are described in U.S. Pat. Nos.5,057,426, 4,923,978, EP Patents 0512767 A1 and EP 0515484B and WO95/13368, WO 97/10331 and WO 96/18731. These patents and patentapplications disclose methods of adsorbing nucleic acids on to a solidsupport and then isolating the nucleic acids. The previously usedmethods use some type of solvent to isolate the nucleic acids and thesesolvents are often flammable, combustible or toxic.

EP0707077A2 describes a synthetic water soluble polymer to precipitatenucleic acids at acid pH and release at alkaline pH. The re-dissolvingof the nucleic acids is performed at extremes of pH, temperature and/orhigh salt concentrations where the nucleic acids, especially RNA, canbecome denatured, degraded or require further purification oradjustments before storage and analysis.

WO 96/09116 discloses mixed mode resins for recovering a targetcompound, especially a protein, from aqueous solution at high or lowionic strength, using changes in pH. The resins have a hydrophobiccharacter at the pH of binding of the target compound and a hydrophilicand/or electrostatic character at the pH of desorption of the targetcompound.

Blood is one of the most abundant sample sources for DNA analysis asblood samples are routinely taken for a wide range of reasons. Howeverbecause of the viscous and proteinaceous nature of blood using known DNAextraction methods it has proved difficult to accomplish usingautomation due to the difficulties of handling large volumes containingrelatively small amounts of DNA. Hitherto nucleic acid extraction hasbeen partially automated only by using hazardous reagents and slowprocessing times.

I have now devised an improved method for the extraction of nucleicacids and other biomolecules from blood and other biological materials,and other samples containing nucleic acid

According to the invention there is provided a method for the extractionof biomolecules from biological material which method comprisescontacting the biological material with a solid phase which is able tobind the biomolecules to it at a first pH and then extracting thebiomolecules bound to the solid phase by elution using an elutionsolvent at a second pH.

In particular there is provided a method for extracting nucleic acidfrom a sample containing nucleic acid, which method comprises:contacting the sample with said solid phase at a first pH at which thesolid phase has a positive charge and will bind negatively chargednucleic acid; and then releasing the nucleic acid at a higher pH atwhich the solid phase possesses a neutral, negative or less positivecharge than at the first pH.

Generally the solid phase will possess an overall positive charge, thatis the sum of all positive and negative charges on the solid phase as awhole is positive. It is possible (though not preferred), however, thatthe solid phase as a whole could be negatively charged, but have areasof predominantly positive charge to which the nucleic acid can bind.Such solid phases are within the scope of the invention.

The change in the charge of the solid phase is referred to herein as“charge switching” and is accomplished by the use of a “charge switchmaterial” in, on or as the solid phase.

The charge switch material comprises an ionisable group, which changescharge to according to the ambient conditions. The charge switchmaterial is chosen so that the pKa of the ionisable group is appropriateto the conditions at which it is desired to bind nucleic acid to andrelease nucleic acid from the solid phase. Generally, nucleic acid willbe bound to the charge switch material at a pH below or roughly equal tothe pKa, when the charge switch material is positively charged, and willbe released at a higher pH (usually above the pKa), when the chargeswitch material is less positively charged, neutral, or negativelycharged.

The present invention is more particularly directed to the use of chargeswitch materials which allow binding and/or releasing (especiallyreleasing) of the nucleic acid to occur under mild conditions oftemperature and/or pH and/or ionic strength.

Generally the charge switch material will change charge because of achange in charge on a positively ionisable group from positive to lesspositive or neutral, as the pH is increased in a range spanning or closeto the pKa of the positively ionisable group. This may also be combinedwith a change of charge on a negatively ionisable group from neutral orless negative to more negative. In an alternative embodiment (describedbelow), however, the charge switch material comprises a material whichis positively charged at both pH values (such as a metal oxide) and anegatively ionisable group, the charge of which becomes more negative asthe pH is increased in a range spanning or close to its pKa.

The charge switch material may comprise an ionisable group having a pKabetween about 3 and 9. For positively ionisable groups, the pKa is morepreferably at least about 4.5, 5.0, 5.5, 6.0 or 6.5 and/or at most about8.5, 9.0, 7.5 or 7.0. A particularly preferred pKa for a positivelyionisable group is between about 5 and 8; even more preferred is a pKabetween about 6.0 and 7.0, more preferably between about 6.5 and 7.0.The pKa for negatively ionisable groups is preferably between about 3and 7, still more preferably between about 4 and 6, further preferablyapproximately at the pH at which it is desired to bind nucleic acid.

Materials having more than one pKa value (e.g. having differentionisable groups), or combinations of materials having different pKavalues, may also be suitable for use as charge switch materials inaccordance with the invention, provided that at a first (lower) pH thematerial(s) possess(es) a positive charge and that at a higher pH thecharge is less positive, neutral or negative.

Generally a charge switch will be achieved by changing the pH from avalue below to a value above the pKa of the or an ionisable group.However, it will be appreciated that when the pH is the same as the pKavalue of a S particular ionisable group, 50% of the individual ionisablegroups will be charged and 50% neutral. Therefore, charge switch effectscan also be achieved by changing the pH in a range close to, but notspanning, the pKa of an ionisable group. For example, at the pKa of anegatively ionisable group, such as a carboxy group (pKa typicallyaround 4), 50% of such groups will be in the ionised form (e.g. COO⁻)and 50% in the neutral form (e.g. COOH). As the pH increases, anincreasing proportion of the groups will be in the negative form.

Preferably the binding step is carried out at a pH of below the pKa ofthe ionisable group, or (though this is not preferred) within about 1 pHunit above the pKa. Generally the releasing step is carried out at a pHabove the pKa of the ionisable group, preferably at a pH between 1 and 3pH units above the pKa.

Prior art methods, such as those disclosed in EP0707077, often use highpH to release the nucleic acid, for example using strong bases such asNaOH. Such high pH can cause depurination of nucleic acid, leading tothe problems of imperfect replication, which can impede subsequent useof the nucleic acid, e.g. in detection and/or amplification techniquessuch as Southern or northern blotting or PCR.

The use of strong bases, or weak bases in combination with heating,again as in EP0707077, can also lead to degradation of RNA (especiallyat pH values of 10 or above), and denaturation of double stranded DNA(i.e. irreversible conversion of DNA from the double stranded form atleast partially into the single stranded form), which can lead to a lackof specific binding in PCR.

The appropriate choice of pKa value(s) in accordance with the inventionallows the step of releasing DNA from the solid phase to be performedunder mild conditions, unlike in the prior art. As used herein, the term“mild conditions” generally means conditions under which nucleic acid isnot denatured and/or not degraded and/or not depurinated, and/orconditions which are substantially physiological.

Preferably the releasing step is performed at a pH of no greater thanabout pH 10.5, more preferably no greater than about pH 10.0, 9.8, 9.6,9.4, 9.2, 9.0, 8.9, 8.8, 8.7, 8.6 or 8.5. Depending on the pKa(s) of thecharge switch material, the releasing step may even be performed atlower pH values, such as 8.0, 7.5 or 7.0. Preferably the releasing stepis carried out in the substantial absence of NaOH, preferably also thesubstantial absence of other alkali metal hydroxides, more preferablythe substantial absence of strong mineral bases. Substantial absence maymean that the concentration is less than 25 mM, preferably less than 20mM, more preferably less than 15 mM or 10 mM.

The desired change in pH can be achieved by altering the ionic strengthof the solution and/or the temperature, since pH is dependent on boththese factors. However, neither high temperature nor high ionic strengthare generally compatible with the desired mild conditions, andaccordingly, the change in pH is preferably not achieved by largechanges in ionic strength or temperature. Moreover, increasing ionicstrength increases competition of charged species with the nucleic acidfor binding to the solid phase, so can assist in releasing the nucleicacid. Small changes of ionic strength are therefore acceptable and maybe used in conjunction with the change in pH to release the nucleicacid, preferably within the limits and ranges given below.

Preferably the temperature at which the releasing step performed is nogreater than about 70° C., more preferably no greater than about 65° C.,60° C., 55° C., 50° C., 45° C. or 40° C. More preferably, suchtemperatures apply to the entire process. The releasing step, or theentire process, may even be performed at lower temperatures, such as 35°C., 30° C. or 25° C.

Furthermore, the releasing step preferably occurs under conditions oflow ionic strength, suitably less than 1M or 500 mM, preferably lessthan 400 mM, 300 mM, 200 mM, 100 mM, 75 mM, 50 mM, 40 mM, 30 mM, 25 mM,20 mM or 15 mM. It may even be below 10 mM. The ionic strength may be atleast about 5 mM, more preferably at least about 10 mM. More preferably,these ionic strengths also apply to the binding step.

PCR is sensitive to pH and the presence of charged contaminants. Inparticularly preferred embodiments, the releasing step is performedusing reagents suitable for storing nucleic acid (such as a commerciallyavailable storage buffer, e.g. 10 mM Tris.HCl, pH8.0-8.5, optionally inthe presence of 1 mM EDTA), or using reagents suitable for use in aprocedure to which the nucleic acid is to be subjected (such as a PCRbuffer, e.g. 10 mM Tris.HCl, 50 mM KCl, pH 8.5).

Common previously known nucleic acid extraction processes require a stepof diluting the elution product containing nucleic acid, to make thesolution suitable for e.g. PCR. Preferably the present inventionsubstantially avoids diluting the released nucleic acid.

Preferably the step of binding DNA occurs under mild conditions,suitably at a pH of no less than 3.0, preferably no less than 3.5, 4.0,4.5 or 5.0. Previous methods have used high concentrations of chaotropicagents, such as 8M guanidine. Such conditions may not be necessary inthe practice of the present invention, in which the binding steppreferably occurs in solution having a total concentration of 2M orless. More preferred temperatures and ionic strengths are as detailedabove for the releasing step.

The use of such mild conditions for the release of nucleic acid isespecially useful for extracting small quantities of nucleic acid, asthe extracted DNA or RNA can be added directly to a reaction or storagetube without further purification steps (e.g. steps necessitated by theuse of high ion concentrations in prior art methods), and without theneed to dilute high ionic strength (as is the case with prior artmethods using high ionic strength to elute the nucleic acid). Thereforeloss of nucleic acid through changing the container, imperfect recoveryduring purification steps, degradation, or denaturation, and dilution ofsmall amounts of nucleic acid can be avoided. This is particularlyadvantageous when a nucleic acid of interest is present in a sample (oris expected to be present) at a low copy number, such as in certaindetection and/or amplification methods.

Broadly speaking, preferred chemical species for use as charge switchmaterials in accordance with the invention comprise a positivelyionisable nitrogen atom, and at least one, but preferably more than one,electronegative group (such as a hydroxy, carboxy, carbonyl, phosphateor sulphonic acid group) or double bond (e.g. C═C double bond), which issufficiently close to the nitrogen atom to lower its pKa. It has beenfound that such molecules tend to have suitable pKa values for theextraction of nucleic acid under mild conditions according to thepresent invention. Preferably at least one (but more preferably morethan one) electronegative group is separated from the ionisable nitrogenby no more than two atoms (usually carbon atoms). Hydroxyl groups areparticularly preferred electronegative groups (particularly when severalhydroxyl groups are present, e.g. in polyhydroxylamines, such as Tris(C(CH₂OH)₃—NH₂) or Bis-Tris (see below)), as they (1) lower the pKa ofthe nitrogen atom (e.g. amine group, e.g. from about 10 or 11) to asuitable value around neutral (i.e. pKa of about 7), (2) allow thespecies to remain soluble/hydrophilic above the pKa, when the nitrogenatom of the amine group loses its positive charge, (3) provide a sitefor covalent linkage to a solid substrate, e.g. a polycarboxylatedpolymer (such as polyacrylic acid), and (4) are uncharged at pH valuessuitable for the releasing step and at which procedures such as PCR areperformed (typically pH 8.5); the presence of charged species caninterfere with PCR especially. Especially preferred are chemical specieshaving an ionisable nitrogen atom and at least 2, 3, 4, 5 or 6 hydroxylgroups.

Many standard, weakly basic, buffers are ideal chemical species toprovide the ionisable groups of charge switch materials, as they havepKa values close to neutral (i.e. 7).

For use as a charge switch material, chemical species comprisingionisable groups can be immobilised onto solid supports (e.g. beads,particles, tubes, wells, probes, dipsticks, pipette tips, slides,fibers, membranes, papers, celluloses, agaroses, glass or plastics) in amonomeric or polymeric form via adsorption, ionic or covalentinteractions, or by covalent attachment to a polymer backbone which isin turn immobilised onto the solid support. Alternatively, they can beincorporated into solid, insoluble forms (with or without attachment toa polymer backbone) which inherently exhibit charge switching, e.g.beads, particles, tubes, wells, probes, dipsticks, pipette tips, slides,fibers, membranes or plastics.

Solid phase materials, especially beads and particles, may bemagnetisable, magnetic or paramagnetic. This can aid removal of thesolid phase from a solution containing the released nucleic acid, priorto further processing or storage of the nucleic acid.

Preferably the weakly basic buffers are biological buffers, i.e. buffersfrom the class of buffers commonly used in biological buffer solutions.Examples of biological buffers may be found in commercial chemicalcatalogues, such as the Sigma catalogue.

Leaching (i.e. transfer from the solid phase into solution in the liquidphase) of chemical species used to provide ionisable groups in ionexchange resins is a virtually inevitable phenomenon to some extent,especially when the species are attached to the solid phase byadsorption. Such leaching typically causes impurity in the resultantproduct, which can lead to significant problems, particularly if theresultant product is intended to be used in PCR (and especially when thespecies are charged). The use of biological buffers to provide theionisable groups in charge switch materials can avoid this problem,since leaching of such buffers into the liquid phase will generally notsignificantly affect the nucleic acid, nor any downstream processes suchas PCR to which it might be subjected. Indeed, many biological buffersare routinely used in PCR buffers, storage buffers and other buffersolutions.

In a particularly preferred embodiment, the releasing step takes placein a buffer solution containing the same biological buffer that is usedin, as or on the charge switch material.

Examples of suitable biological buffers for use in charge switchmaterials in accordance with the invention, and their pica values, areas follows:

-   -   N-2-acetamido-2-aminoethanesulfonic acid ‡‡ (ACES), pica 6.8;    -   N-2-acetamido-2-iminodiacetic acid ‡‡ (ADA), pica 6.6; amino        methyl propanediol † (AMP), pKa 8.8;    -   3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic        acid † (AMPSO), pKa 9.0;    -   N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid †† (BES), pica        7.1;    -   N,N-bis-2-hydroxyethylglycine † (BICINE), pKa 8.3;    -   bis-2-hydroxyethyliminotrishydroxymethylmethane ‡‡ (Bis-Tris),        pKa 6.5;    -   1,3-bistrishydroxymethylmethylaminopropane ‡‡ (BIS-TRIS        Propane), pKa 6.8;    -   4-cyclohexylamino-1-butane sulfonic acid (CABS), pKa 10.7;    -   3-cyclohexylamino-1-propane sulfonic acid (CAPS), pKa 10.4;    -   3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), pKa        9.6;    -   2-N-cyclohexylaminoethanesulfonic acid (CHES) pKa 9.6;    -   3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid ††        (DIPSO), pKa 7.6;    -   N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid †† (EPPS or        HEPPS), pKa 8.0;    -   N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid † (HEPBS),        pKa 8.3;    -   N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid †† (HEPES),        pKa 7.5;    -   N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid †† (HEPPSO),        pKa 7.8;    -   2-N-morpholinoethanesulfonic acid ‡ (MES), pKa 6.1;    -   4-N-morpholinobutanesulfonic acid †† (MOSS), pKa 7.6;    -   3-N-morpholinopropanesulfonic acid †† (MOPS), pKa 7.2;    -   3-N-morpholino-2-hydroxypropanesulfonic acid ‡‡ (MOPSO), pKa        6.9;    -   piperazine-N—N-bis-2-ethanesulfonic acid ‡‡ (PIPES), pKa 6.8;    -   piperazine-N—N-bis-2-hydroxypropanesulfonic acid †† (POPSO), pKa        7.8;    -   N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid † (TABS),        pKA 8.9;    -   N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid ††        (TAPS), pKa 8.4;    -   3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid        †† (TAPSO), pKa 7.4;    -   N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid †† (TES),        pKa 7.4;    -   N-trishydroxymethylmethylglycine † (TRICINE), pKa 8.1; and    -   trishydroxymethylaminomethane † (TRIS), pKa 8.1,    -   histidine*, pKa 6.0, and polyhistidine ‡‡;    -   imidazole*, pKa 6.9, and derivatives* thereof (i.e. imidazoles),        especially derivatives containing hydroxyl groups**;    -   triethanolamine dimers**, oligomers** and polymers**; and    -   di/tri/oligo amino acids**, for example Gly-Gly, pKa 8.2; and        Ser-Ser, Gly-Gly-Gly, and Ser-Gly, the latter three having pKa        values in the range 7-9.

In a preferred embodiment, the buffers marked above with an asterisk (*)are not considered to be biological buffers for the purposes of theinvention (whether or not they are designated as such in any chemicalcatalogue). In a more preferred embodiment, those marked with twoasterisks (**) are also not considered to be biological buffers.Preferred biological buffers are marked with a dagger (†), morepreferred buffers are marked with two daggers (††), still more preferredbuffers are marked with a double dagger (‡) and most preferred buffersare marked with two double daggers (‡‡).

These and other chemical species comprising ionisable groups may becoated as monomers onto a solid phase support using covalent, ionic oradsorption interactions. Additionally or alternatively, they may becoated onto such solid phase supports in polymeric form (preferablyfollowing condensation polymerisation), for example by adsorption onto anegatively charged surface (e.g. a surface having exposed COOH or SO₃groups), or by covalent attachment. Additionally or alternatively, thechemical species containing ionisable groups may be attached to apolymer (see below) which is then attached to a solid support, e.g. byadsorption or covalent attachment.

Preferably the chemical species or polymer backbones are covalentlycoupled to the solid support via a hydroxyl group or other group so thatthe ionisable group having the desired pKa value (usually, but notlimited to, a nitrogen atom) remains capable of binding and releasingnucleic acid.

Biological buffers and other chemical species comprising positivelyionisable groups may be used in conjunction with a chemical speciescontaining a negatively ionisable group which has a suitable pKa,preferably in the ranges described above. For example a biologicalbuffer (having one or more positively ionisable nitrogen atoms) may beattached to a polymer or other solid phase material which has exposedcarboxy groups even after attachment of the biological buffer. Such amaterial may bind nucleic acids at a low pH when few of the carboxygroups are negatively charged (i.e. few are in the COO⁻ form, most beingin the COOH form) and most of the ionisable nitrogen atoms arepositively charged. At higher pH the negative charge is stronger (i.e. agreater proportion of carboxy groups are in the COO⁻ form) and/or thepositive charge is weaker, and the nucleic acid is repelled from thesolid phase.

Chemical species containing ionisable groups (such as the biologicalbuffers listed above) can be attached to a polymer backbone using knownchemistries. For example a chemical species containing a hydroxyl groupcan be attached using carbodiimide chemistry to a carboxylated polymerbackbones. Other chemistries include can be employed by someone skilledin the art using other polymer backbones (e.g. based on polyethyleneglycol (PEG) or carbohydrate) using a range of standard couplingchemistries (see e.g. Immobilised Affinity Ligand Techniques, Greg T.Hermanson, A. Krishna Mallia and Paul K. Smith, Academic Press, Inc.,San Diego, Calif., 1992, ISBN 0123423309, which is incorporated hereinby reference in its entirety.)

Alternatively, the chemical species containing ionisable groups can bepolymerised without a backbone polymer, using cross-linking agents, forexample reagents that couple via a hydroxy group (e.g.carbonyldiimidazole, butanediol diglycidyl ether, dialdehydes,diisothiocyanates). Polymers may also be formed by simple condensationchemistries to generate polymeric amino acids with the appropriate pKae.g. Gly-Gly.

Preferably such immobilisation, attachment and/or polymerisation of thechemical species containing the ionisable group does not affect the pKaof the ionisable group, or leaves it in the desired ranges given above.For example it is generally preferred not to couple or polymerise thechemical species via a positively ionisable nitrogen atom (in contrastfor example to WO97/2982). In the practice of the invention, it isespecially preferred to immobilise, attach and/or polymerise thechemical species via an hydroxyl group.

A preferred polymeric material is a dimer or oligomer of Bis-Tris, or amaterial formed by attaching a plurality of Bis-Tris molecules to apolyacrylic acid backbone, e.g. by reacting Bis-Tris monomer withpolyacrylic acid using 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC).The polymer can then be easily separated from the reactants usingdialysis against a suitable reagent or water. Preferably the polyacrylicacid has molecular weight of between about 500 and 5 million or more.More preferably it has a molecular weight of between 100,000 and500,000.

The nature of the resultant Bis-Tris/polyacrylic acid molecule willdepend on the ratio of the coupled components, since the polymer willhave different properties depending on the proportion of the acrylicacid groups that are modified with Bis-Tris, for example it is desirablefor some carboxy groups to remain unmodified, as the presence of thesewill not prevent the Bis-Tris from binding nucleic acid at low pH(especially if the Bis-Tris is in excess), but their negative charge athigher pHs will assist with release of the nucleic acid. For use in thepresent invention, the molar ratio of Bis-Tris:carboxy groups (beforeattachment) is preferably between 5:1 and 1:5, more preferably between3:1 and 1:3, still more preferably between 2:1 and 1:2, furtherpreferably between 1.5:1 and 1:1.5, and most preferably about 1:1.

The presence of high residual charge (i.e. charged species present insolution along with the extracted nucleic acid) may adversely affect theanalysis of nucleic acids by PCR, or interfere with the binding ofprimers, dNTPs or polymerase to the nucleic acid, or to thesequestration of Mg²⁺ ions, which are essential to PCR. It isparticularly preferable to avoid residual positive charge.

Preferred materials for use in the invention, such as the biologicalbuffers described above, possess minimal residual positive charge(preferably minimal residual charge) at the pH at which the nucleic acidis released, and/or at pHs 8-8.5, making interference with or inhibitionof downstream processes unlikely.

Patent application PCT/GB00/02211, of the same inventor, disclosescertain methods within the scope of the present invention and isincorporated herein by reference in its entirety as exemplification ofthe present invention (in all its aspects—see below for other aspects ofthe invention). In particular, it discloses a method for the extractionof biomolecules from biological material which method comprisescontacting the biological material with a solid phase which incorporateshistidine or a polyhistidine which will tend to bind nucleic acids at Slow pH and then extracting the biomolecules bound to the solid phase byelution using an elution solvent which will then release the boundnucleic acids when the pH is increased.

An alternative embodiment of the present invention uses a material whichis positively charged across a wide pH range, such as 0-12 or 0-14 (e.g.an electropositive substance such as a metal oxide, metal, strong orweak base, which lacks a pKa value, or for which the pKa value is at anextreme of high pH. Such a positively charged material is combined withnegatively ionisable material having a pKa intermediate between the pHvalues at which it is desired to bind and release nucleic acid, orslightly below the pH at which it is desired to bind nucleic acid. Thiscombination of materials allows nucleic acid to be bound at certain pHvalues, around and below the pKa of the negatively ionizable material,when there are fewer negatively charged groups, but allows the nucleicacid to be released when the pH is increased and a greater number of theionisable groups are negatively charged. For example, the combination ofiron II,III oxide and polycarboxylates (see Examples) binds nucleic acidat pH 4, when a relative scarcity of negative charges allowing thepositively charged iron oxides to bind the nucleic acid. When the pH isincreased to around 8, a large proportion of the carboxy groups becomenegatively charged and, despite the remaining presence of positivecharges on the iron oxides, the reduction in overall positive chargeallows the nucleic acid to be released.

Further examples of charge switching molecules for nucleic acidpurification are based on detergents or surfactants that have ahydrophobic portion and a hydrophilic portion which comprises apositively ionisable group with a suitable pKa, e.g. decyl methylimidazole or dodecyl-Bis-Tris. These detergents/surfactants can beadsorbed onto surfaces e.g. plastic via their hydrophobic portions andthe hydrophilic (ionisable) portions can be used to capture nucleicacid.

Another family of suitable materials for capture and easy release ofnucleic acids are carbohydrates e.g. glucosamine, polyglucosamine(including chitosans), kanamycins and their derivatives i.e. sugar ringbased structures containing one or more nitrogen atoms surrounded byhydroxyl groups which may also contain other groups such as acetate orsulphate groups to provide a suitable pKa for binding and release ofnucleic acids.

Another group of materials with suitable pKa values are nucleic acidbases, e.g. cytidine (pKa 4.2). The can be immobilised via hydroxygroups to a polymer or solid phase carboxy group using carbodiimides.

A still further group of materials having members with suitable pKavalues are heterocyclic nitrogen-containing compounds. Such compoundsmay be aromatic or aliphatic and may be monomers, oligomers or polymers,such as morpholine-, pyrrole-, pyrrolidine-, pyridine-, pyridinol-,pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-,piperidine-, or piperazine-containing compounds, e.g. polyvinylpyridine.Such compounds may be substituted with electronegative groups to bringthe pKa value(s) of the ionisable nitrogen atom(s) into an acceptablerange, e.g. as defined above. However, in some compounds this may not benecessary, the pKa already being in such a range.

Preferred materials for use in accordance with the invention arehydrophilic, for example comprising charge switch materials which are(or which comprise chemical species which before immobilisation orpolymerisation are) water soluble.

Once a suitable solid phase has been prepared, comprising a chargeswitch material, repeated capture and release of nucleic acids can beperformed by adjusting the pH up or down. Thus sequential reactions oranalysis can be performed on the nucleic acids using the same solidphase. For example, DNA can be isolated from a biological sample using aPCR tube comprising a charge switch material. Then, following PCR, theamplified DNA product may be isolated from the buffer constituents orprimers by adjusting the pH in the same tube.

Particularly preferred solid phase materials are non-porous. Poroussupports are commonly used for isolating proteins, which can be trappedin the pores of the support. However, nucleic acids tend to be too bigto enter into pores of commonly used such supports, and will thereforebecome bound to the surface of the support, potentially trappingimpurities in the pores.

The method can be used to separate single stranded RNA or DNA fromdouble stranded DNA, because of the different charge densities on singleand double stranded molecules, by appropriate manipulation of the pH orsalt concentration. Typically, single stranded molecules will bereleased from binding to the solid phase at a lower pH than doublestranded molecules.

In some circumstances, for example for the construction of gene chips,and for the preparation of probes, it may be desirable to produce singlestranded DNA. Manipulation of pH and/or ionic strength can assist inpurification and release of single stranded nucleic acid. The method ofthe invention may comprise a prior step of converting double strandednucleic acid in the sample to single stranded nucleic acid (preferablyusing a strong base, e.g. 100 mM NaOH, or a weak base at hightemperature, e.g. 60-100° C.). The solid phase material is preferablythen added simultaneously with a buffer which changes the pH of thesample to the pH for binding single stranded nucleic acid (typically apH of 4-7).

The materials described herein may also be employed to capture nucleicacids in the liquid phase where binding leads to a cross-linked latticelarge enough to separated from the liquid phase, e.g. by filtration orcentrifugation.

Accordingly, in a second aspect, the present invention provides a methodfor extracting nucleic acid from a sample containing nucleic acids,which method comprises: contacting the sample with a charge switchmaterial at a first pH at which the charge switch material has apositive charge and will bind negatively charged nucleic acid; and thenreleasing the nucleic acid at a second, higher pH at which the charge isneutral, negative or less positive than at the first pH, wherein thecharge switch material is soluble at said first pH, and wherein thecombination of the charge switch material and the bound nucleic acid isinsoluble at or above said first pH and below said second pH.

Preferred features of the method are as set out above, with theexception of the charge switch material being formed into, immobilisedon, or attached to, a solid phase material.

Usually the charge switch materials will be soluble at the second pH,and will remain in solution with the nucleic acid upon release of thenucleic acid; the use of a weakly basic buffer (optionally bound to asoluble backbone, e.g. polyacrylic acid) as the charge switch materialcan avoid problems of contamination as described above.

The methods of the invention preferably include one or more washingsteps between the binding and releasing steps. Such (a) washing step(s)will generally be carried out at said first pH, or a pH above said firstpH but lower then said second pH, such that the nucleic acid issubstantially not released during the washing step(s).

As has been indicated previously, the methods of the invention areparticularly suitable for extracting nucleic acid which is then storedor further processed (e.g. by PCR), particularly when the charge switchmaterial is in the form of e.g. a tube or well in which such storageand/or processing can occur. For the avoidance of doubt, however, it isemphasised that the releasing step and any subsequent storage orprocessing need not be carried out as discrete steps, but can coincide,when said storage or processing occurs at a pH at which release of thenucleic acid occurs. For example, the method of the invention includesbinding nucleic acid to a charge switch material coated on or otherwiseprovided by a PCR tube, washing the bound nucleic acid, and then withouta separate releasing step commencing the PCR reaction using a PCR bufferwhich causes release of the nucleic acid.

In a further aspect, the present invention provides novel charge switchmaterials for use in the methods of the receding aspects. It furthercomprises the use of such charge switch materials in such methods. Allpreferred features of the charge switch materials described in above inthe context of the methods apply equally and independently to thepresent aspect of the invention (i.e. preferred combinations of featuresmay be different in relation to this aspect from the preferredcombinations in relation to the method aspects).

In a further aspect, the present invention provides a container(preferably a PCR or storage tube or well, or a pipette tip) coatedwith, comprising or formed from a charge switch material, preferably acharge switch material comprising a biological buffer.

The following description is directed particularly to the extraction ofnucleic acid from blood, but applies also to the extraction of nucleicacid from any liquid sample, particularly biological samples or samplesproduced during laboratory techniques, such as PCR.

The method is particularly useful if the biological material is blood,but the method can be used for a range of applications substances suchas plasmid and vector isolation and plant DNA extraction.

Preferably the cells in the blood are lysed to release nucleic acids andknown lysing agents and methods can be used, such as contacting withionic and non ionic detergents, hypotonic solutions of salts, proteases,chaotropic agents, solvents, using pH changes or heat. A method oflysing cells to isolate nucleic acid is described in WO 96/00228.

When the biological material consists of blood the samples canoptionally be diluted with water or other diluent in order to make iteasier to manipulate and to process.

Dilutions up to ten times can be used and in general more dilution canbe better and it is a feature of the present invention that it allowslow dilution of blood to be possible.

The solid phase with which the blood is contacted, can be a formed of amaterial which has a natural affinity for nucleic acids or it can beformed of a material which has its surface treated with an agent whichwill cause nucleic acids to bind to it or increase its affinity fornucleic acids. Suitable materials include controlled pore glass,polysaccharide (agarose or cellulose), other types of silica/glass,ceramic materials, porous plastic materials such as porous plastic plugswhich in a single moulded part or as an insert in a standard tube,polystyrene beads para magnetic beads etc. The size and porosity is notcritical and can vary and be selected for particular applications.

Suitable means for treating the surface of the solid phase or forderivatising it include treating it with a substance which can introducea charge e.g. a positive charge on the surface or a hydrophilic orhydrophobic surface on the solid phase e.g. hydroxyl groups, nitrategroups, autoreactive groups, dyes and other aromatic compounds.

In a preferred embodiment of the invention the solid phase will causeDNA to be bound to it at one pH in preference to contaminants in theblood sample and will allow the bound nucleic acid to be released whenit is contacted with an eluant at a different pH. This system can beused with a solid phase which incorporates histidine or a polyhistidinewhich will tend to bind nucleic acids at low pH e.g. less than 6 andwill then release the bound nucleic acids when the pH is increased e.g.to greater than 8. Alternatively the nucleic acids are bound atsubstantially neutral pH to an aminated surface and released at veryhigh pH.

In another embodiment of the invention a plastic moulding canincorporate a binding agent e.g. in a well in a plate etc. so that thebinding agent is incorporated in the surface, the blood sample is thencontacted with the surface so as to cause nucleic acids to be bound tothe surface. The blood sample is then removed and the surface treatedwith an eluting agent to release the bound nucleic acids. When thesurface is part of a well in a multi-well plate, the total system can bereadily adapted for rapid large scale sampling and extractiontechniques.

Binding agents which can be used include charge switchable ion exchangeresins using a positively charged solid phase that can be reversed ormade neutral by changing the pH above its pKa. e.g. nucleotides,polyamines, imidazole groups and other similar reagents with a suitablepKa value.

Also, nucleic acids can be bound by intercalation using a variety ofintercalating compounds incorporated into the solid phase e.g.actinomycin D, ethidium bromide etc.

In a further embodiment of the invention a plastic surface can bemodified to include functional groups. The plastic can be any plasticused for containing samples e.g. polypropylene. The functional groupscan be positively or negatively charged so as to bind the nucleic acidsin the correct buffer solution.

Alternatively the functional groups can be chemical groups capable ofcovalent coupling to other ligands or polymers.

When the plastic is used in a plastic moulding e.g. in a well in aplate, or as a polymerase chain reaction (PCR) tube, the surfacecharacteristics of the plastic can be suitably modified for use in thepresent invention by including or adding the appropriate chemicals inthe moulding compound e.g. as in an injection moulding compound.

When this is used in a PCR tube or in a deep well plate the tubes orwells can be used to isolate and immobilise small quantities of DNA orRNA generating a pure template for subsequent PCR or other geneticanalysis and manipulation.

When the plastic is polypropylene e.g. it is in the form of a thinwalled PCR tube the polypropylene surface can be modified by oxidisingthe surface with an oxidising agent such as potassium permanganate andsulphuric acid to create a carboxylated surface (COOH groups). This tubecan then be used to improve the isolation of DNA from solutions or fromcrude samples e.g. blood. By adjusting the pH, di-electric constant,solubility or ionic strength the DNA or RNA can be immobilised on thewalls of the tube, washed free of contaminants, ready for PCR or otheranalytical techniques.

The carboxy groups can be further modified by covalently coupling ananionic group such as imidazole or polyhistidine or any strong or weakion exchanger, to allow binding of nucleic acids by a chargeinteraction. This tube could then be used to improve the isolation ofDNA from solutions or from crude samples e.g. of blood. Again byadjusting the pH, di-electric constant, or ionic strength the DNA or RNAcan be immobilised on the walls of the tube, washed free ofcontaminants, ready for PCR or other analytical techniques.

The nucleic acids can be eluted with in a low salt buffer so that it isready for PCR or other analysis.

The solid phase can be contacted with a blood sample by mixing with thesolid phase in a mixing/stirring device, by passing the blood sampleover the solid phase or the solid phase can be paramagnetic andmanipulated by a magnetic field. Although the invention is particularlysuitable for the separation or isolation of nucleic acids from blood itcan be used with a range of biomolecules particularly those that requireremoval of cell wall debris or insoluble particles.

In a preferred embodiment of the invention the solid phase is ingranular form in a column and the blood sample is drawn up through thecolumn by means of a pressure differential being applied through thecolumn, the blood sample is drawn up with air and the granular solidmaterial can become fluidised thus increasing the mixing and contactingrates and minimising clogging.

The method of the invention is suitable for use in a multi-well formatwhen a series of extractions from different samples can take placesubstantially simultaneously and this will facilitate the automation ofthe extraction process allowing rapid high throughput extraction to takeplace and to allow combinational chemistry to be performed. This willenable there to be a high throughput in a standard well array e.g. aneight by twelve array so that a large number of sample types can betreated automatically at the same time.

The invention, in its various aspects, will now be described in detail,by way of example only.

EXAMPLE 1 Extraction of Nucleic Acids from Whole Blood

A charge switchable ion-exchanger was prepared by covalently couplingpolyhistidine to 100 (m glass beads using glutaldehyde by mixing 1 gramof the aminated glass beads with 0.01% (v/v) glutaldehyde in 0.1M sodiumbicarbonate at pH8 containing 20 mg polyhistidine. After overnightincubation the beads were washed exhaustively to remove non-covalentlybound material and stored in 10 mM MES, pH5 containing 0.1% (v/v) Tween20.

About 300 mg of the 100 (m derivitised glass beads were added to a 1 mlplastic column enclosed at both ends.

A blood sample was incubated with an equal volume of 10 mM MES pH5,containing 1% Tween 20, proteases (200 (g/ml) and 1 mM EDTA. Afterdigestion is complete the blood was sucked up the column containing theglass beads and the DNA became immobilised allowing the contaminatingproteins to pass through to waste.

The glass beads containing the immobilised DNA were washed with a buffercomprising 10 mM MES pH5, containing 1% Tween 20, and 1 mM EDTA and thiswas repeated until the wash solution was colourless.

After washing, the beads were dried with air and DNA eluted with a smallquantity of 10 mM Tris HCl, pH 8.5 and collected in a sterile tube readyfor analysis. Thus the DNA were separated from the blood.

For different biomolecules, the buffer etc. can be suitably modified.

EXAMPLE 2

One gram of carboxylated paramagnetic beads were washed in 50 mMImidazole buffer pH6 and then mixed with 100 mg of polyhistidine in 50ml of 50 mM Imidazole buffer pH 6. A chemical coupling agent was added(EDC) at a final concentration of 5 mg per ml and mixed overnight. Thebeads were washed in water, 0.5M sodium chloride, 1% Tween 20, 100 mMTris HCl pH 8 and stored in 10 mM MES, 0.1% Tween 20 pH5.

To extract DNA from blood, 1 mg of beads were mixed with blood dilutedin 10% Tween 20 with 25 mM MES, 1 mM EDTA pH 5. The beads were separatedwith a magnet and washed by resuspending in 1 mM MES, 0.1% Tween 20. Toelute the DNA the beads were resuspended in 10 mM Tris HCl pH 8.5 andseparated with magnet leaving the DNA in solution.

EXAMPLE 3 Bis-Tris Solid Phase Magnetic Beads

200 mg of carboxylated 1 μm magnetic particles were reacted in a onestep procedure with 100 mg of Bis-Tris and 100 mg of the carbodiimide,EDC, in 50 mM imidazole buffer pH6.0. Following an overnight incubation,the magnetic particles were washed and used to isolate Plasmid DNA.

An alkaline lysis method was used to prepare a cleared 5 ml bacteriallysate generating a supernatant containing the plasmid in 0.5M potassiumacetate, pH5. To the supernatant, 2.5 mg of magnetic particles wereadded and mixed for 1 minute. After magnetic separation and washing withwater pH5, the pure plasmid DNA was eluted off in 200 μl of 10 mMTris.HCl pH 8.5.

The magnetic beads were also used to extract DNA directly from wholeblood using a detergent based digestion reagent containing proteinase K.

EXAMPLE 4 Tricine on Solid Phase Magnetic Beads

50 mg of carboxylated 1 μm magnetic particles were reacted in a one stepprocedure with 50 mg of Tricine and 100 mg of the carbodiimide, EDC, in50 mM imidazole buffer pH6.0. Following an overnight incubation, themagnetic particles were washed and used to isolate Plasmid DNA. Analkaline lysis method was used to prepare a cleared 5 ml bacteriallysate generating a supernatant containing the plasmid in 0.5M potassiumacetate, pH5. To the supernatant, 2.5 mg of magnetic particles wereadded and mixed for 1 minute. After magnetic separation and washing withwater pH5, the pure nucleic acids were eluted off in 200 μl of 10 mMTris.HCl pH 6.5.

EXAMPLE 5 Bis-Tris Solid Phase Polystyrene Beads

1 gram of carboxylated 60 μm polystyrene particles were reacted in a onestep procedure with 500 mg of Bis-Tris and 500 mg of the carbodiimide,EDC, in 50 mM imidazole buffer pH6.0. Following an overnight incubation,the particles were washed and used to isolate plasmid nucleic acids asdescribed above.

EXAMPLE 6 Bis-Tris Polymer

Bis-Tris monomer was converted into a polymer by mixing together 160 mgof polyacrylic acid with a molecular weight of 240,000, 1.6 g ofBis-Tris and 1.6 g of EDC in 50 mM imidazole pH6.0. Following anovernight incubation, the mixture was dialysed in water. The purifiedpolymer was then coated onto magnetic COOK beads or used in the liquidphase to bind genomic DNA from blood. A 5 ml blood sample wascentrifuged to obtain the nuclei and WBC population and the resultingpellet digested with 1% SOS. Following precipitation with potassiumacetate the cleared supernatant was mixed with either 25 mg ofmagnetic-Bis-Tris or about 250 μg of poly-Bis-Tris as a liquid. In bothcases the captured DNA could be separated, washed in water and thenredissolved in 10 mM Tris HCl pH8.5 in a pure form.

EXAMPLE 7 Insoluble Tris HCl Polymer

In this example an insoluble polymer was made with inherent chargeswitching properties by mixing 80 mg of polyacrylic acid with 800 mg ofTris HCl and 800 mg of EDC in 50 mM Imidazole pH6. The insolubleprecipitate that formed generated a particulate solid phase that wasused to capture DNA and release it in a similar manner to that describedin example 4 for genomic DNA.

EXAMPLE 8 Immobilised Poly Bis-Tris on Tips

A solution of poly Bis-Tris at 1 mg/ml, prepared as in Example 2, in0.1M sodium bicarbonate pH8 incubated at 60° C. for 8 hours with twenty200 μl polyproplylene pipette tips. The tips were then rinsed and usedto capture about 150 ng of plasmid DNA from a cleared bacterial lysateby pumping up and down ten times. After a quick wash with water pH5, theDNA was eluted in 50 μl of 10 mM Tris pH 8.5.

EXAMPLE 9 Immobilised Poly Bis-Tris on PCR Tubes

A solution of poly Bis-Tris at 1 mg/ml, prepared as in Example 2, in0.1M sodium bicarbonate pH8 incubated at 60° C. for 8 hours in a 200 μlPCR plate of 8×12 tubes. After rinsing, the tubes were used to bindgenomic DNA from a sample prepared according to example 4. About 50 ngof DNA was subsequently eluted off per tube using 10 mM Tris HCl pH8.5.

EXAMPLE 10 Charge Switch Detergents in Liquid Phase

A blood sample was prepared as described in Example 4 and to theresulting supernatant decyl imidazole was added at pH 4 causingprecipitation of the DNA. The DNA pellet was collected by centrifugationand redissolved in 10 mM Tris pH 8.5.

EXAMPLE 11 Charge Switch Detergents on Solid Phase

Decyl imidazole was adsorbed onto a 200 μl plastic pipette tip bysoaking in a 1% solution at pH4 in 0.1M sodium acetate. A blood samplewas prepared as described in Example 3 and the tips were used to bindthe DNA by repeated pumping and sucking. After a wash with water, about50 ng of DNA was recovered in water at pH10.

EXAMPLE 12 Polyglucosamines

10 mg of low molecular weight Chitosan was dissolved in acidified waterand then 50 mM imidazole pH5.5, this was mixed with 100 mg of carboxy 1μm magnetic beads and with 20 mg of the carbodiimide EDC in 50 mMimidazole pH5.5. Following an overnight incubation, the beads werewashed and resuspended in 10 mM MES pH5. To bind genomic DNA, 2 mg ofmagnetic particles were added to a supernatant prepared by methodsdescribed earlier in Example 1, after magnetic separation, the DNA waseluted using 100 mM Tris.HCl pH 9.5.

EXAMPLE 13 Kanamycin

A solution of genomic DNA was prepared as described in example 3. Tothis sample 2 mg of Kanmycin was added at a concentration of 10 mg/ml.The resulting precipitate of DNA was filtered, washed in water at pH5and re-dissolved in water at pH10.

EXAMPLE 14 Magnetizable Iron Oxides in Carboxylated Polystyrene

A 5 ml Plasmid mini-prep was prepared using standard alkaline lysisreagents to generate a cleared lysate with a potassium acetatecomposition of 0.5M pH4. To this cleared supernatant, 2.5 mg ofcommercially available 1 μm carboxylated polystyrene magnetisableparticles were added to bind the plasmid DNA. The particles were washedwith water at pH4 and then the DNA eluted using 10 mM Tris HCl at pH8.5. Typical UV ratios at 260 and 280 nm were 1.7-2.0, indicating purenucleic acids with a single band observed with standard gelelectrophoresis.

EXAMPLE 15 Titanium Dioxide in Polystyrene Microtitre Plates

A solution of DNA at 100 μg per ml in 0.1M Potassium Acetate pH4 wasallowed to stand for 1 hour in a 300 μl flat bottomed microtitre plasticplate, the plastic plate contained titanium oxide which was incorporatedas a powder in the plastic when the plate was formed. After washing atpH4, the DNA was recovered with water at pH10 and 2 ml measured at 260nm versus a plain polystyrene plate with no titanium oxide.Approximately, 50 ng of DNA was recovered per 300 μl well for the plateincorporating the titanium oxide compared to zero for the plainpolystyrene plate.

EXAMPLE 16 Cytidine Coupled to Magnetic Beads

1 gram of carboxylated 1 μm magnetic particles were reacted in a onestep procedure with 500 mg of Cytidine and 500 mg of the carbodiimide,EDC, in 50 mM imidazole buffer pH6.0. After thorough washing, the beadswere used to bind nucleic acids from a plasmid preparation as describedin example 1 and recovering the pure nucleic acids in water at pH10.

EXAMPLE 17 Polyvinyl Pyridine (PVP)

20 mg of commercially available PVP beads was mixed with the supernatantcontaining genomic DNA from a 5 ml blood extraction described in example4. After allowing the DNA to bind, the beads were washed with water atpH5 and the DNA recovered using water at pH10. Ultra violet analysis at260 and 200 nm indicated a purity ratio of 1.65.

EXAMPLE 18 Separation of RNA and DNA

A solution of tRNA and sheared genomic DNA was prepared at 30 μg per mlin 50 mM Potassium acetate buffer pH6.5 with 1M sodium chloride.Approximately 4 mg of magnetic polyhistidine beads were mixed with 1 mlof the nucleic acid solution for one minute until binding was complete.The beads were then thoroughly washed with water at pH5. To elute thebound material, the beads were mixed with 300 μl of 10 mM Tris.HCl, 10mM NaCl, pH8.5. Gel analysis showed that most of the tRNA remained insolution and was not bound to the beads. The eluted material containedmostly genomic DNA with little or no tRNA.

EXAMPLE 19 DNA Analysis

In all previous examples, extracted DNA was analysed by one or more ofthe following:

-   -   (1): ultra violet (UV) analysis at 260 nm and 280 nm, to provide        a measure of nucleic acid concentration;    -   (2): Gel electrophoresis using 1% agarose in TBE buffer run at        60V for 20 minutes vs a commercial preparation of DNA as        control, with ethidium bromide staining to measure molecular        size and to provide an estimate of quantity of the nucleic acid;        or    -   (3): PCR using primers specific for actin or other ubiquitous        genes, to test integrity of the nucleic acid.

The results are presented as (1): direct readings from the instrument;and (2): and (3): gel pictures.

In all cases, the examples demonstrated effective extraction of nucleicacid which was not significantly damaged.

1-33. (canceled)
 34. A water soluble product for use in a method ofextracting nucleic acid from a sample, the product comprising aplurality of positively ionisable groups, the ionisable groups beingprovided by a chemical species selected from the list consisting of:biological buffers; polyhydroxylated amines; histidine; andpolyhistidine.
 35. A product according to claim 34 wherein thebiological buffer is selected from the group consisting of:N-2-acetamido-2-amlnoethanesulfonic acid (ACES);N-2-acetamido-2-iminodiacetic acid (ADA); amino methyl propanediol(AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid(AMPSO); N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);N,N-bis-2-hydroxyethylglycine (BICINE);bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);N,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);4-cyclohexylamino-1-butane sulfonic acid (CABS);3-cyclohexylamino-1-propane sulfonic acid (CAPS);3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);2-N-cyclohexylaminoethanesulfonic acid (CHES);3-N,N-bis-2-hydroxyethylamino-2 hydroxypropanesulfonic acid (DIPSO);N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);2-N-morpholinoethanesulfonic acid (MES); 4-N-morpholinobutanesulfonicacid (MOBS); 3-N-morpholinopropanesulfonic acid (MOPS);3-N-morpholino-2˜hydroxypropanesulfonic acid (MOPSO);piperazine-N—N-bis-2-ethanesulfonic acid (PIPES);piperazine-N—N-bis-2-hydroxypropanesulfonic acid (POPSO);N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS);N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);3-N-trishydroxymethyl-methylamino-2hydroxypropanesulfonic acid (TAPSO);N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);N-trishydroxymethylmethylglycine (TRICINE);trishydroxymethylaminomethane (Tris); polyhistidine; polyhydroxylatedimidazoles; triethanolamine dimers and polymers; and di/tri/oligo aminoacids, for example Gly-Gly, Ser-Serf Gly-Gly-Gly, and Ser-Gly.
 36. Aproduct according to claim 34, wherein the plurality of ionisable groupsare separately attached to a polymer.
 37. A product according to claim34, wherein the ionisable groups are polymerised, optionally by means ofcross-linking reagents.
 38. A product for use in a method of extractingnucleic acid from a sample, wherein the product possesses a positivecharge at both a first pH at which it is desired to bind nucleic acidand a second higher pH at which it is desired to release nucleic acid,the product comprising a plurality of negatively ionisable groups, thecombined charge of which becomes more negative between said first pH andsaid second pH, such that the product is capable of binding nucleic acidat said first pH, which bound nucleic acid is released from the productat said second pH.
 39. A product according to claim 38, wherein thenegatively ionisable group has a pKa between about 3 and 7, preferablybetween about 4 and
 7. 40. A product according to claim 38; wherein thenegatively ionisable is a carboxy group.
 41. A product according toclaim 38 wherein said positive charge is provided by a metal or metaloxide, preferably iron II,III oxide.
 42. A method for extracting nucleicacid from a sample containing nucleic acid, which method comprises: at afirst pH, bringing the sample into contact with a material in whichchemical species are immobilised on a solid phase, the chemical specieshaving a positively ionisable nitrogen atom and at least two hydroxylgroups, wherein at least one of the hydroxyl groups is separated fromthe ionisable nitrogen by no more than two atoms, wherein the materialhas a positive charge at said first pH, such that nucleic acid is boundto the material; and releasing the nucleic acid at a second, higher, pHat which the charge on the material is negative, neutral or lesspositive, wherein the positively ionisable nitrogen atom has a pKabetween 4.5 and 8.5 and the release of the nucleic acid occurs undermild conditions.
 43. The method according to claim 42, wherein aplurality of the chemical species providing the ionisable groups areseparately immobilised on a solid phase by covalent or ionic bonding orby adsorption.
 44. The method according to claim 42, wherein a pluralityof the chemical species providing the ionisable groups are separatelyattached to a polymer, said polymer being immobilised on a solid phaseby covalent or ionic bonding or by adsorption.
 45. The method accordingto claim 44, wherein the chemical species are covalently linked to acarboxylated polymer.
 46. The method according to claim 42, wherein thechemical species providing the ionisable groups are polymerised,optionally by means of cross-linking reagents.
 47. The method accordingto claim 46, wherein the polymer is immobilised on a solid support bycovalent or ionic bonding or by adsorption.
 48. The method according toclaim 42, wherein the chemical species providing the ionisable group isselected from amino methyl propanediol (AMP),N,N-bis-2-hydroxyethylglycine (BICINE),bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris),1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane),N-trishydroxymethylmethylglycine (TRICINE) ortrishydroxymethylaminomethane (Tris).
 49. The method according to anyone of claims 42, wherein the chemical species is a dialcohol aminereagent or a carbohydrate.
 50. The method according to claim 49, whereinthe dialcohol amine is diethanol amine or the carbohydrate isglucosamine, polyglucosamine, chitosan or kanamycin.
 51. The methodaccording to claim 42, wherein the chemical species is represented bythe general formula [HO—(CH₂)_(n)]₂—N—(CH₂)_(m), wherein n and m andintegers between 1 and 10, and is attached to the solid phase using asilane reagent.
 52. The method according to claim 51, wherein thechemical species is 3-bis(2-hydroxyethyl)aminopropyltriethoxy silane.53. The method according to claims 42, wherein the chemical speciesproviding the ionisable groups is a dimer or oligomer of Bis-Tris orcomprises a plurality of Bis-Tris molecules attached to a polyacrylicacid backbone.